Clostridium botulinum is a spore forming, obligate anaerobic bacterium that naturally resides in soil. All C. botulinum subgroups produce botulinum neurotoxins (BoNTs), which generally act to impair synaptic transmission and are effective in curtailing uncontrolled muscle contractions. Accordingly, botulinum neurotoxins have many important therapeutic, medical, and cosmetic applications.
BoNTs act by cleaving and inactivating proteins that function in neurotransmitter release at synapses. Neurotransmitter release involves exocytosis, whereby synaptic vesicles filled with neurotransmitter fuse with the plasma membrane of a nerve terminal. This process is mediated by the assembly of a synaptic fusion complex, which is comprised of a set of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNARE proteins). BoNTs target three different SNARE proteins that perform functions essential for membrane fusion and successful neurotransmitter release at the synapse: syntaxin, SNAP-25 and synaptobrevin.
The seven different BoNT serotypes are designated by the letters A through G. In particular, BoNT/C1, which has a structure similar but not identical to other BoNT serotypes, is composed of a 100 kD heavy chain and a 50 kD light chain. The heavy chain binds to the surface of a cell by recognizing a receptor. After binding, the light chain translocates across the membrane and enters the cell. The light chain contains a zinc dependent endopeptidase which is responsible for the catalytic cleavage of the target SNARE proteins.
BoNT/A and BoNT/E cleave SNAP-25, while BoNT/B, BoNT/D, BoNT/F, and BoNT/G cleave synaptobrevin. BoNT/C1 is unique in that it cleaves both SNAP-25 and syntaxin; thus it is the only known BoNT that cleaves syntaxin, and it is the only known BoNT having dual substrate specificity. BoNT/C1 cleaves SNAP-25 at the bond between Arg198 and Ala199 (one residue before the BoNT/A cleavage site between Gln197 and Arg198), and syntaxin at the bond between Lys253 and Ala254. There are two isoforms of syntaxin, designated 1A and 1B, and BoNT/C1 cleaves them both at the same site.
Although there are BoNTs that specifically cleave synaptobrevin and SNAP-25, none of the naturally occurring BoNTs cleave only syntaxin. A protein that targets syntaxin while not affecting SNAP-25 or other SNARE protein activity would be a powerful research tool for neurophysiologists studying the role of specific proteins such as syntaxin in neurotransmission.
Furthermore, recently published research suggests that fluctuating levels of syntaxin may be correlated to glucose intolerance and diabetes, as well as possibly being associated with a broad range of neurological disorders, such as Williams Syndrome, schizophrenia, and severe depression (Lam, P. P. L., Y. Leung, L. Sheu, J. Ellis, R. G. Tsuchima, L. R. Osborne, and H. Y. Gaisano, Diabetes (2005) 54, 2744-2754). In addition, other research has indicated that fluctuations of syntaxin levels in the myocardium may affect the therapeutic effectiveness of ATP sensitive potassium channel openers (Ng, B., Y. Kang, H. Xie, H. Sun, and H. Gaisano, Cardiovascular Research (2008) 80, 365-374). Thus, an entity that specifically targets syntaxin for cleavage while not affecting other biological targets could potentially have utility as a therapeutic agent.